LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

The Open PCR machines is a DYI device that is composed of many circuit boards, wires, and a wooden frame. It is to be used to cycle DNA by oscillating the temperature of the DNA samples. This machine predominately works when the samples are placed in the main heating block, at which point a heated lid is placed down on top of the samples. Once the software for this Open PCR device is set up, the temperature change and the actual process begins. Within the Open PCR machine is a multitude of parts that keep the machine intact. These parts include a heat sink and fan to absorb heat, a circuit board that runs all the parts, a power supply to maintain the electricity, and a LCD display to show the user information. In conclusion, all of these parts work cohesively to generate this working machine known as the Open PCR.

Experimenting With the Connections
It is important to note that the LCD display will not work if it is not connected to the Open PCR circuit Board. Also, the temperature will not be shown on the LCD display if the white wire connecting to the main heating block is disconnected from the Open PCR circuit board.

Test Run
The test run was done November 1st, 2012. Machine number 12 was used and there were minimal problems. It felt as if the machine was running slower than it should, but other than that all went well.

Protocols

Polymerase Chain Reaction

A polymerase chain reaction (PCR) is based on the enzyme DNA Polymerase's ability to synthesize complementary DNA strands. Through a series of steps involving polymerase breaking apart a DNA strand and then synthesizing a specified complementary piece, a PCR machine is able to isolate and amplify a desired strand of DNA.

Steps to Amplify a Patient's DNA Sample

1. PCR uses controlled temperature changes to make copies of DNA. Heat (about 95°C) separates double-stranded DNA into two single strands; this process is called denaturation.

2. "Primers", or short DNA strands, binds to the very end of the complimentary sequence that is being replicated. This step is called annealing, which takes place between 40°C and 65°C. The temperature that we used was 57°C.

3. Once the annealing process is done, the temperature is raised to about 72°C and DNA polymerase then extends from the primers copying the DNA.

4. PCR then amplifies a segment of a DNA sequence. In the end, there will be two new DNA strands identical to the original strand.

Components of PCR Master Mix

• A modified form of the enzyme Taq DNA polymerase that lacks 5´→3´ exonuclease activity.

• dNTPs

• MgCl2

• Colorless Reaction Buffer (pH 8.5)

Components of PCR Master Mix

Reagent

Volume

Template DNA (20 ng)

0.2μL

10μM forward primer

1.0μL

10μM reverse primer

1.0μL

GoTaq master mix

50.0μL

dH2O

47.8μL

Total Volume

100μL

Sample 1:
Patient ID: 11014
Age: 67
Gender: Male
Replicate: 1

Sample 2:
Patient ID: 11014
Age: 67
Gender: Male
Replicate: 2

Sample 3:
Patient ID: 11014
Age: 67
Gender: Male
Replicate: 3

Sample 4:
Patient ID: 46446
Age: 62
Gender: Female
Replicate: 1

Sample 5:
Patient ID: 46446
Age: 62
Gender: Female
Replicate: 2

Sample 6:
Patient ID: 46446
Age: 62
Gender: Female
Replicate: 3

Sample 7:
Positive Control

Sample 8:
Negative Control

Flourimeter Assembly Procedure

1. To assemble the flourimeter, first obtain smartphone to capture the picture needed during data collection.

2. Turn on the flourimeter and drop a single drop of solution onto the hydrophobic slide.

3. Turn the black box provided upside down to cover the flourimeter.

4. Set up the smartphone on the stand provided, and align the camera/phone about 3 inches in front of the flourimeter. Make sure that the stand and the flourimeter is covered directly under the black box.

Proper assembly of the flourimeter.

Steps To Prepare Samples For The Flourimeter

You will have 8 samples from the OpenPCR instrument and 1 DNA (calf thymus standard at 2 micrograms/mL) sample and water from the scintillation vial to analyze.

1. With a permanent marker, number your transfer pipettes at the bulbs so that you only use if for one sample. With the permanent marker number your Eppendorf tubes at the top. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled.

2. Transfer each sample seperatly (using one pipette per sample) into an Eppendorf tube containing 400 mL of buffer. Label this tube with the number of your sample. Get your entire sample into this Eppendorf tube. You can use this sample number transfer pipette to place only this sample drop onto the fluorescent measuring device.

3. Take the specially labeled Eppendorf tube containing Sybr Green I using the specifically labeled pipette only place two drops on the first two centered drops as seen on the video.

4. Now take your diluted sample and place two drops on top of the Syber Green I solution drops.

5. Align the light going through the drop, as seen in the video.

6. Let the smart-phone operator take as many pictures using the light box as he/she wants.

7. Now either rerun the sample again or discard that sample’s pipette. Keep the Sybr Green I labeled pipette.

8. You can run 5 samples per glass slide.

9. As the last sample run the water from the scintillation vial as a blank using the same procedure as with the other samples.

Procedure for Capturing Images of the Flourimeter with a Smartphone

Our group used a Galaxy Nexus

1. After setting up the Flourimeter set a Smartphone’s photo settings to the ones listed.

Inactivate the flash

Set ISO To 800 (or higher if possible)

Set White Balance to Auto

Set Exposure to Highest Setting

Set Saturation to the Highest Setting

Set Contrast to Lowest Setting

2. Once the samples have been prepared, place the Flourimeter in the light box.

3. Take as many pictures as needed. Your goal is to take pictures clear enough so ImageJ can take data from the images.

4. Once you have taken enough photos of that sample give the Flourimeter back to the sample preparer to prepare the next sample.

5. Repeat this procedure for all the samples.

A pure drop of water on the hydrophobic slide

Procedure for Opening Images in ImageJ

1. ImageJ was used to analyze the images taken by the smartphone. To upload the image onto ImageJ, the ANALYZE tab was clicked and SET MEASUREMENTS was chosen. AREA INTEGRATED DENSITY and MEAN GREY VALUE was selected from the menu.

2. The MENU tab was selected and COLOR was chosen, the function SPLIT CHANNELS was used; three separate files were created. SYBR GREEN fluoresces green, so the image name with "green" next to it was used.

3. The oval selection was used to draw an oval around the green drop. Then, MEASURE was selected from the ANALYZE tab, and the sample number and the numbers measured from the image were recorded.

4. To get the readings from the background of the image, another oval of approximately the same size was drawn in the background green image and MEASURE was selected from ANALYZE tab. The sample number and the numbers measured from the image was recorded, this data will be labeled as "background".

5. The measurements were saved in an excel file by clicking SAVED AS from the FILE tab.

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

PCR detection works by heating the DNA sample to about 110°C in order to split the DNA. Then the PCR cools off to 57°C in order for the primer to attach to the DNA strands. The PCR then heats to 72°C so the DNA strand can be re-written. The rs17879961 cancer-associated sequence will produce a DNA signal because the reverse primer used, AACTCTTACACTCGATACAT(The letters in the sequence are the bases and stand for Guanine (G), Adenine (A), Cytosine (C), and Thymine (T).
) will only attach if the DNA sample has the same coding with the cancer-associated sequence “ACT”. If the DNA sample does not have the cancer-associated sequence the primer will not attach because the sequence is AACTCTTACACTTCGATACAT, and there will be no DNA signal. The primer sequences that will be used is ACTC or in reverse CTCA.

A positive result will be known because there will be a profound amount of the same sequence, the rs17879961. If there is none of the sequence than we know that the results are negative.

Baye's Law (worksheet)

Basically Baye's Rule is that the results are not 100% accurate. Therefore, there will be some incorrect results. False positives and false negatives can occur often. Baye's Rule is what is used to find the false positive and false negative results.
[IMG][/IMG]
Source: http://openpcr.org/use-it/

Results

Sample Name

Background ID

Area

X

Y

W

H

Mean Pixel Value

Raw INTDEN

INTDEN (if different)

Water (drop)

4 drops of Water

40090

900

1617

243

210

86.83

3481003

--

Water (Background)

4 drops of Water

40090

--

--

--

--

11.641

466695

--

Calf Thymus DNA (drop)

2 drops of Sybr Green I & 2 drops of Calf Thymus DNA

23060

765

1587

204

144

178.144

4107996

--

Calf Thymus DNA (background)

2 drops of Sybr Green I & 2 drops of Calf Thymus DNA

21712

--

--

--

--

13.897

301733

--

PCR: Negative Control (drop)

2 drops of Sybr Green I & 2 drops of C-

53988

873

1809

276

249

78.544

4240449

--

PCR: Negative Control (background)

2 drops of Sybr Green I & 2 drops of C-

53988

--

--

--

--

13.293

717685

--

PCR: Positive Control (drop)

2 drops of Sybr Green I & 2 drops of C+

42819

687

1656

249

219

136.836

5859196

--

PCR: Positive Control (background)

2 drops of Sybr Green I & 2 drops of C+

42819

--

--

--

--

11.79

504846

--

PCR: Patient A ID 11014, rep 1 (drop)

2 drops of Sybr Green I & 2 drops of 1A

32840

849

1626

258

165

230.513

7570053

--

PCR: Patient A ID 11014, rep 1 (background)

2 drops of Sybr Green I & 2 drops of 1A

32872

--

--

--

10.493

344912

--

PCR: Patient A ID 11014, rep 2 (drop)

2 drops of Sybr Green I & 2 drops of 2A

24996

780

1674

204

156

194.658

4865678

--

PCR: Patient A ID 11014, rep 2 (background)

2 drops of Sybr Green I & 2 drops of 2A

23568

--

--

--

--

10.886

256551

--

PCR: Patient A ID 11014, rep 3 (drop)

2 drops of Sybr Green I & 2 drops of 3A

34832

852

1743

231

192

232.768

8107782

--

PCR: Patient A ID 11014, rep 3 (background)

2 drops of Sybr Green I & 2 drops of 3A

34356

--

--

--

--

12.878

442441

--

PCR: Patient B ID 46446, rep 1 (drop)

2 drops of Sybr Green I & 2 drops of 1B

62026

687

2013

342

231

173.954

10789693

--

PCR: Patient B ID 46446, rep 1 (background)

2 drops of Sybr Green I & 2 drops of 1B

62872

--

--

--

--

11.930

750043

--

PCR: Patient B ID 46446, rep 2 (drop)

2 drops of Sybr Green I & 2 drops of 2B

46912

789

1797

252

237

162.36

7616625

--

PCR: Patient B ID 46446, rep 2 (background)

2 drops of Sybr Green I & 2 drops of 2B

46265

--

--

--

--

14.646

677576

--

PCR: Patient B ID 46446, rep 3 (drop)

2 drops of Sybr Green I & 2 drops of 3B

40980

771

1734

252

207

122.754

5030472

--

PCR: Patient B ID 46446, rep 3 (background)

2 drops of Sybr Green I & 2 drops of 3B

40980

--

--

--

--

12.530

513464

--

Sample

INTDEN (drop)

INTDEN (background)

INTDEN with Background Subtracted

DNA μg/mL

Conclusion

Water

3491003

466695

3014308

None

Calf Thymus DNA

4107996

301733

3806263

2.0

PCR: Negative Control

4240449

717685

3522764

1.9

PCR: Positive Control

5859196

504846

5354332

2.8

PCR: Patient A ID 11014, rep 1

7570053

344912

7225141

3.8

PCR: Patient A ID 11014, rep 2

4865678

256551

4609127

2.4

PCR: Patient A ID 11014, rep 3

8107782

442441

7665341

4

PCR: Patient B ID 46446, rep 1

10789693

750043

10039650

4.3

PCR: Patient B ID 46446, rep 2

7616625

677576

6939049

3.6

PCR: Patient B ID 46446, rep 3

5030472

513464

4517008

2.4

KEY

Sample = What the contents are of the sample being tested. There were 10 samples total: 8 samples from the OpenPCR instrument, 1 DNA sample and 1 water sample.

Rep = Repeat

Drop = The values obatined when you draw the best oval around the (green) drop image and then select ANALYZE > MEASURE

Background = The values obtained when you draw another oval of the same size in the (green) file for the background above the drop to get the “noise," and select ANALYZE > MEASURE

INTDEN = Integrated Density

Integrated Density = The sum of the values of the pixels in the selected part of the image. Since the image viewed was the green channel, the value of each pixel is the amount of green present in that pixel. We subtracted the INTDEN from the background of the image in order to eliminate any noise and get a more accurate result.

DNA μg/mL = This column of estimated DNA concentrations was calculated using a proportional calibration based on the given values: water's subtracted INTDEN of 3014308 had 0μg/mL and the DNA CALF THYMUS with a subtracted INTDEN value of 3806263 has 2μg/mL of DNA.